In vitro stem cell models Flashcards
What is a unipotent cell?
A cell that can only give rise to ONE specific lineage
What signals does the niche provide the stem cell with?
Signals that promote self-renewal
What happens when the stem exits from the niche?
There is exposure to differentiation signals (these are different to the self-renewal signals
Why are mice the best mammalian system to study developmental biology?
- Takes 20 days to have a new born
- Large litter
- Can be manipulated
Which cells are pluripotent?
Cells found in the very early embryo, that can give rise to ALL the cells of the body, including the germ cells (but not the extra embryonic tissue)
Where are the pluripotent cells found?
Inner cell mass of the blastocyst stage embryo
What are 2 ways to test if a cell is a stem cell?
1) Descriptive test - cell expresses pluripotency factors
2) Functional test - cell causes teratoma formation
Describe the descriptional test of the proposed stem cells
- Stem cells express a SPECIFIC set of pluripotency markers
- Using in situ hybridisation - use to detect the RNA expression of these set of makers SPECIFICALLY CONFINED tot the the inner cell mass (where PS cells are found)
What are the main pluripotency markers)
Nanog
Oct4
Sox2
When do cells remain pluripotent until?
What do these cells express?
They are still pluripotent STRAIGHT after implantation of the mouse embryo into the uterus
Express pluripotency markers: Nanog, Oct4 and Sox2
Describe the functional test of the proposed stem cells
- Graft PS cells (from a mouse embryo straight after implantation) into the kidney of a host mouse
- Cells grafted should give rise to teratocarcinoma (a pluripotent tumour formed of ALL cell types)
Why is the proposed stem cell grafted into the kidney in the functional test?
Permissive environment - provides the right signals for the cell to divide into all the cells of the body
When do the pluripotent cells of the body begin to differentiate?
At GASTRULATION
When does gastrulation occur?
After implantation
What is gastrulation?
A highly morphogenetic process where 1 layer transforms to 3 layer, which are the first founders of all the cells that will make the embryo proper
What does the ectoderm give rise to?
Surface
Neural
Neural crest
(skin, peripheral and central nervous system)
What does the mesoderm give rise o?
Axial
Paraxial
Intermediate
Lateral
(blood, heart, kidney, muscle, bone)
What does the endoderm give rise to?
Gut
Internal organs
(liver, pancreas, intestine)
At what stage in development are the pluripotent cells extinct?
E7/E7.5 (end of gastrulation)
When do the neural stem cells form?
E9 at the future forebrain
What is the potency of neural stem cells?
What cells can they generate?
Bipotent - can generate 2 different stem cell types
Neurons and glia
What do glial cells do?
Surround the neuron
Provide support and nutrients
Why are ES cells hard to study in vivo?
What is the solution to this?
Not very accessible
Small in numbers
In utero development
Ethics
Solution - CAPTURE the stem cells in vitro
What can in vitro modelling of embryonic development be useful for?
- Drug screening
- Cell replacement therapies
What signals must/must not be present to capture the self-renewing state of an ES cell?
- Need to MIMIC the conditions in the NICHE
- Need to STOP the process of differentiation (block using INHIBITORS)
What are the critical signals in the mouse to maintain cells in a self-renewing, undifferentiated state?
LIF (leukaemia inhibitory factor)
BMP antagonists
What are the critical signals in the human to maintain cells in a self-renewing, undifferentiated state?
FGF2
TGF beta
What are the stages of capturing the ES cell in vitro?
1) Take blastocyst stage embryo (E4 in mouse) and DISSOCIATE the inner cell mass using a laser
2) Plate the inner cells on layer of FEEDER cells